The pathology associated with toxoplasmosis stems from the parasite's lytic cycle initiated by active invasion of host cells. The parasite has a complex secretory system essential for host cell invasion and establishment of the parasitophorous vacuole. Biogenesis of these organelles remains poorly understood but several groups have postulated a compartment situated between Golgi and mature micronemes or rhoptries in which sorting and processing occurs. We have identified a new compartment and named it multivesicular endosome (MVE) and in this proposal we will test the hypothesis that this compartment represents the endocytic/exocytotic hub where sorting of proteins targeted to various organelles occurs. We will also test the hypothesis that this MVE present in T. gondii tachyzoites, is linked to the acidocalcisomes since both share a similar proton pump (vacuolar proton pyrophosphatase or VP1) and appear to interact. We know that it is also involved in the microneme pathway, as supported by experiments showing that microneme 2 associated protein (M2AP) traverses this compartment en route to the micronemes. The localization of a cathepsin L (CPL) in this compartment supports its postulated role in processing of M2AP. We also hypothesize that this vacuole is the same or is closely related to the multivesicular endosomal compartment that was shown to accumulate rhoptry protein 2 (ROP2) when its dileucine motifs were mutated to dialanine or when the amino acid residues of the 51 subunit of adaptor protein 1 (AP-1) involved in their recognition, were altered by mutagenesis. This compartment could not be identified before because of lack of adequate markers. In addition to VP1, the MVE possesses a water channel or aquaporin (AQP), a property that it shares with the plant cell vacuole and the contractile vacuole and acidocalcisomes of Trypanosoma cruzi. Our hypotheses are that: (1) the T. gondii MVE belongs to the endosomal system and is an intermediate compartment in the trafficking of proteins from the Golgi complex to acidocalcisomes, and secretory organelles;and (2) the T. gondii multivesicular endosome is involved in cellular homeostatic processes. Our specific aims are: (1) Characterize the T. gondii MVE as a trafficking organelle and (2) To study the role of the T. gondii MVE in osmoregulation and intracellular pH homeostasis. These pilot studies will characterize a new parasite compartment of potential great importance to the parasite invasion and host- subversion process. We will develop robust markers, and generate the reagents necessary for future in depth study aimed at a detailed characterization of the trafficking pathway to other secretory organelles and at understanding the physiological roles of the MVE. xoplasma gondii is a pathogenic protozoan parasite infecting a wide range of vertebrate hosts including humans. In immunocompetent individuals, infection is largely asymptomatic and is controlled by the host's immune response. However, T. gondii has also been recognized as a major opportunistic pathogen of fetuses from recently infected mothers and of immunocompromised patients, i.e. those with AIDS. T. gondii can be transmitted through contaminated food and/or water and because of this, it has been listed by NIH as category B priority pathogen. Our studies on characterization of a novel organelle and the study of trafficking of proteins to acidocalcisomes could reveal novel targets for the chemotherapy of toxoplasmosis. PUBLIC HEALTH RELEVANCE: Our goal is to find ways of interfering with Toxoplasma gondii metabolic pathways as a strategy for controlling infections caused by this and similar parasites. Differences in endocytic traffic between T. gondii and its mammalian host could be exploited for chemotherapeutic purposes. This work is designed to test the hypothesis that a multivesicular endosome present in these parasites is involved in trafficking of proteins to acidocalcisomes, and has a role in osmoregulation and intracellular pH homeostasis.